Abstract Single molecule techniques have fueled key insights into the molecular basis of protein function by facilitating the ability to characterize individual conformational populations and dynamics. In recent years, there has been an explosion in the capabilities of instrumentation, and a vast number of biological questions have been identified that could be addressed using this technique, especially if multiple distances could be simultaneously measured in large protein molecules (requiring 3-6 site specific labels). However, methodologies for generating proteins with the requisite labels have lagged, limiting applications needing multi-site labeling. We propose to develop, adapt and combine a robust set of protein synthesis / semisynthesis methodologies centered around the mechanistically related chemistries of native chemical ligation and protein trans splicing to the challenge of developing novel tools for building multiply-labeled proteins. Through careful synthetic design, we have developed a strategy that will facilitate site-specific protein labeling at multiple positions in a protein using a single, commonly-practiced labeling reaction. This semisynthetic approach is compatible with any protein, regardless of length or sequence and has the potential to close the gap between experiments that can be imagined and those that can be performed in the laboratory.